Ambient outdoor air pollutants and sex ratio of singletons born after in vitro fertilization: the effect of single blastocyst transfer.

OBJECTIVE: To evaluate the impact of air pollution on the sex ratio in singletons after IVF treatment and to evaluate the influence of the number of and the developmental stage of transferred embryos on the sex ratio. DESIGN: Retrospective cohort study. SETTING: University-affiliated IVF unit. PATIENT(S): A total of 7,004 singletons born after fresh transfer or frozen-thawed embryo transfer (FET) between January 2013 and December 2017. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Male-to-female ratio in live-born singletons. RESULT(S): The estimated medians (interquartile range) of particle matter (PM)10, PM2.5, CO, NO2, O3, and SO2 at the IVF site were 51.4 (39.5-64.6), 27.7 (20.7-37.4), 0.62 (0.5-0.72), 32.5 (25.4-40.1), 79.6 (63.3-96.6), and 11.9 (9.3-15.9) µg/m3, respectively. Multivariate analysis indicated that SO2 was the only pollutant clearly associated with sex ratio. In singletons from single blastocyst transfer (SBT), as indicated by the generalized additive model, the SO2 concentration and sex ratio showed an inverted-U-shape association. In singletons after non-SBT, a monotonic decreasing in the sex ratio was observed with increased SO2 concentration. Compared with the referent category (SO2 < 7.57 µg/m3), the sex ratio at the 5th decile of SO2 (10.81-11.94 µg/m3) was increased by 2.1-fold (95% confidence interval [CI], 1.3-3.14) after adjusting covariates. In singletons born from non-SBT, the sex ratio significantly decreased only in the 9th (odds ratio = 0.69; 95% CI, 0.53-0.90) and 10th (OR = 0.74, 95% CI, 0.56-0.98) deciles. CONCLUSION(S): Low concentrations of SO2 showed an association with increased sex ratio in singletons of SBT, while in singletons born from another ET system the sex ratios did not show an association at low concentrations of SO2.

Association between outdoor air pollution during in vitro culture and the outcomes of frozen-thawed embryo transfer.

STUDY QUESTION: Does outdoor air pollution differentially affect the outcomes of frozen-thawed embryo transfer (FET) and fresh transfer in IVF treatment? SUMMARY ANSWER: Increased SO2 and O3 levels at the site of IVF unit were significantly associated with lower live birth rates following FET but did not affect the contemporary fresh transfer outcomes. WHAT IS KNOWN ALREADY: Ambient air pollution has been associated with human infertility and IVF outcomes. However, most of the studies excluded FET cycles. STUDY DESIGN, SIZE, DURATION: A retrospective cohort study of 11148 patients contributing to 16290 transfer cycles between January 2013 and December 2016 was carried out. PARTICIPANTS/MATERIALS, SETTING, METHODS: The average age of the cohort was 31.51 ± 4.48 years and the average BMI was 21.14 ± 2.37 kg/cm2. Inverse distance weighting interpolation was used to estimate the daily ambient exposures to six pollutants (PM2.5, PM10, SO2, NO2, CO, O3) at an IVF clinical site, according to the data from fixed air quality monitoring stations in the city. The exposures of each cycle were presented as average daily concentrations of pollutants from oocyte retrieval to embryo transfer/cryopreservation. Exposures were analyzed in quartiles. A generalized estimating equation was used to evaluate the association between pollutants and IVF outcomes, adjusted for important confounding factors including maternal age, infertility diagnosis, BMI, endometrial status and embryo transfer policy. MAIN RESULTS AND THE ROLE OF CHANCE: The clinical pregnancy rate and live birth rate of the cycles was 55.1% (8981/16290) and 47.1% (7672/16290), respectively. Among the included cycles, 4013 patients received 5299 FET cycles, resulting in 2263 live births (42.7% per ET), whereas 9553 patients received 10991 fresh transfer cycles, resulting in 5409 live births (49.2% per ET). SO2 and O3 levels were significantly associated with live birth rates in FET cycles, whereas none of the pollutants were significantly associated with IVF outcomes in contemporary fresh transfer cycles. The FET cycles in the highest quartile of SO2 and O3 exposure had significantly lower live birth rates (adjusted odds ratio (OR) 0.63, 95%CI 0.53-0.74; 0.69, 95% CI 0.58-0.82, respectively) in comparison with those in the lowest quartile. Models involving all transfer cycles and interaction terms (FET×exposures) suggested that FET significantly enhanced the effects of SO2 and O3 exposure on IVF outcomes (P < 0.001). Multi-pollutant models gave consistent results for the association between SO2 and live birth in FET cycles. Accounting for all six pollutants, women in the highest quartile of SO2 still had the lowest live birth rates (OR 0.61, 95%CI 0.47-0.80). LIMITATIONS, REASONS FOR CAUTION: The study was limited by its retrospective nature. The exposure data were estimated according to monitoring data rather than measured directly from the IVF unit. Unknown confounding factors may skew the results. WIDER IMPLICATIONS OF THE FINDINGS: Our data implied that embryos undergoing FET may be more vulnerable to a suboptimal environment than those undergoing fresh transfer. In heavily polluted sites or seasons, fluctuation in FET outcomes may be partially explained by the dynamic changes of ambient gaseous air pollutant. STUDY FUNDING/COMPETING INTEREST(S): National Natural Science Foundation (81302454). The authors have no conflicts of interest to declare. TRIAL REGISTRATION NUMBER: N/A.

Day 6 blastocyst is associated with increased birth weight in full-term singleton newborns after frozen-thawed transfer.

PURPOSE: The purpose of the study is to compare the newborns weight in singleton term birth following transfer of thawed blastocysts-frozen on either day 5 or day 6 after in vitro fertilization. METHOD: The retrospective study included 1444 frozen-thawed blastocyst transfer (FBT) cycles resulting in live singleton births between Jan 2013 and Dec 2016. The main outcomes measured were absolute birth weight, z-score adjusted for gestational age and gender, and incidence of large-for-gestational-age (LGA) newborns. Generalized linear model (GLM) and logistic regression were used in multivariate analyses. RESULT(S): Both the absolute birth weight (3416.49 ± 404.74 vs 3349.22 ± 416.17) and the z-score (0.6 ± 0.93 vs 0.41 ± 0.93) were significantly higher on day 6 FBT in comparison with day 5 FBT. The incidence of LGA newborns was also increased on day 6 FBT (22.8 vs 14.7%, P = 0.006). Adjusted for maternal age, BMI, PCOS diagnosis, present of vanishing twin, and embryo quality, the odds ratio (95% confidence interval) for LGA on day 6 FBT comparing with day 5 FBT was 1.76 (1.18-2.64). CONCLUSION(S): Day 6 FBT is associated with increased birth weight and contributes to the incidence of LGA newborns in FBT.

Differential response of AMH to GnRH agonist among individuals: the effect on ovarian stimulation outcomes.

PURPOSE: The purpose of this study is to investigate whether individual response of anti-Mullerian hormone (AMH) to gonadotropin-releasing hormone (GnRH) treatment is associated with difference in ovarian stimulation outcomes. METHODS: The retrospective study included 1058 non-polycystic ovary syndrome (PCOS) women undergoing long agonist protocol in a single in vitro fertilization unit from January 1, 2016, through December 31, 2016. Patients were grouped according to AMH changes from day 3 to the day of stimulation (group 1, change < 1 ng/ml, n = 714; group 2, decrease ≥ 1 ng/ml, n = 143; group 3, increase ≥ 1 ng/ml, n = 201). A generalized linear model including Poisson distribution and log link function was used to evaluate the association between AMH response and the number of oocytes retrieved. RESULTS: Group 2 was characterized by higher basal AMH level and increased AMH to AFC ratio in comparison with two other groups. However, the number of oocytes and ovarian sensitivity index in group 2 was significantly lower than group 3. Adjusted for age, BMI, ovarian reserve markers, and stimulation parameters, the population marginal means (95% confidence interval) of oocyte number in groups 1 through 3 were 9.51 (9.17, 9.86), 8.04 (7.54, 8.58), and 10.65 (10.15, 11.18), respectively. For patients from group 2 and group 3, basal AMH is no longer significantly associated with oocyte yield. CONCLUSIONS: AMH change in response to GnRH agonist varies among individuals; for those undergoing significant changes in AMH following GnRH agonist treatment, basal AMH may not be a reliable marker for ovarian response in long agonist protocol.

Low body mass index compromises live birth rate in fresh transfer in vitro fertilization cycles: a retrospective study in a Chinese population.

OBJECTIVE: To evaluate the effects of low body mass index (BMI) on in vitro fertilization (IVF) outcomes in fresh transfer cycles. DESIGN: Retrospective cohort study. SETTING: University-affiliated hospital. PATIENT(S): A total of 4,798 cycles with conventional stimulation and fresh transfer in a single IVF center during the period 2013-2014. Low BMI (<18.5 kg/m2) was defined according to World Health Organization guidelines, and cycles within a normal weight range (18.5-24.9 kg/m2) were used as reference. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Live birth rate per fresh embryo transfer. RESULT(S): Low BMI was associated with reduced live birth rates and increased miscarriage rates compared with normal weight, controlling for important covariates known to influence IVF outcomes. Patient age was the most potent confounder, causing a 10.5% reduction in the odds ratio (OR) for live birth between the groups compared. When an interaction term (age × BMI) was introduced, the OR for live birth was reduced in cycles of those aged ≥35 years compared with cycles of those aged 28-34 years, whereas the change in OR between cycles in those aged <28 and cycles in those aged 28-34 years was insignificant. CONCLUSION(S): Low BMI is associated with negative outcomes in fresh transfer cycles, especially for women of advanced age.

Polychlorinated biphenyls impair endometrial receptivity in vitro via regulating mir-30d expression and epithelial mesenchymal transition.

Polychlorinated biphenyls (PCBs) are ubiquitous legacy persistent pollutants and epidemiological data showed that PCB burdens were associated with failed implantation in human. However, the mechanism how PCB exposure affects the embryo implantation is not clear. Using an in vitro model for human embryo implantation employing the human choriocarcinoma cell line JAR and the human endometrial cell line Ishikawa, we have shown that PCB mixture Aroclor 1254 at environmental-relevant concentrations (2.5, 12.5, and 62.5µM) dose-dependently impaired the endometrial receptivity by reducing the adhesion of JAR spheroid attachment and increasing the spheroid outgrowth. The receptive-up-regulated micro-RNA, mir-30d was also down-regulated in endometrial cells by the exposure. Following transient transfection of mir-30d mimic, the disrupted attachment and outgrowth of JAR spheroids was partially restored in the model. By measurement of cadherin switch and vimentin expression, the PCB exposure also activated epithelial mesenchymal transition (EMT) in endometrial cells. In accordance, mir-30d mimic suppressed the EMT markers induced by PCBs. Luciferase reporter assay confirmed that the EMT regulator Snai1 was targeted by mir-30d, and the expression of Snai1 was dose-dependently up-regulated by PCB exposure. Taken together, our study revealed that PCBs may affect the receptivity of endometrial cells by impairing the interaction between receptivity-up-regulated microRNA and EMT process.

Effects of previous ovarian drilling on cumulative ongoing pregnancy rates among patients with polycystic ovarian syndrome undergoing in vitro fertilization.

OBJECTIVE: To determine if history of undergoing laparoscopic ovarian drilling (LOD) was associated with changes in cumulative ongoing pregnancy rates following in-vitro fertilization in patients with polycystic ovary syndrome (PCOS). METHODS: The present retrospective study was performed in an in vitro fertilization unit in Xiamen, China, between June 1, 2008 and December 31, 2012. Patient characteristics and in vitro fertilization treatment outcomes were compared between patients with PCOS and a history of LOD (LOD group), patients with PCOS without a history of LOD (no-LOD group), and age-matched patients without PCOS and with no history of LOD (age-matched group). RESULTS: The study included 110 patients in the LOD group, 127 patients in the no-LOD group, and 990 patients in the age-matched group. A lower number of retrieved oocytes, fewer available embryos, and a lower number of cryopreserved embryos were observed in among patients in the LOD-group compared with the other groups (P≤0.001). No differences in birth rates following fresh embryo transfers were observed between the LOD-group, and the age-matched group (P=0.274) and the no-LOD group (P=0.120). A higher adjusted odds ratio of cumulative pregnancy per initiated in vitro fertilization cycle was observed in the no-LOD group when compared with the LOD group (1.976; 95% confidence interval 1.091-3.580). CONCLUSION: LOD could compromise cumulative ongoing pregnancy rates during subsequent in vitro fertilization.

Non-coding RNA LINC00473 mediates decidualization of human endometrial stromal cells in response to cAMP signaling.

Decidualization is an essential step in the establishment of pregnancy. However, the functional contributions of long intergenic noncoding RNAs (LincRNAs) to decidualization have not been explored. To explore the regulation and role of LincRNAs during human decidualization, human endometrial stromal cells (HESCs) are induced to undergo in vitro decidualization by treating with estradiol-17ß, db-cAMP and medroxyprogesterone acetate. LINC00473 (LINC473) expression is highly induced in HESCs after decidual stimulus. We found that cAMP-PKA pathway regulates the expression of LINC473 through IL-11-mediated STAT3 phosphorylation. RNA interference-mediated down-regulation of LINC473 inhibits in vitro decidualization. These results suggested that LINC473 might be functionally required for human decidualization. This is the first report demonstrating the presence of LincRNA during human decidualization.

Decrease in prosaposin in spermatozoon is associated with polychlorinated biphenyl exposure.

Polychlorinated biphenyls (PCBs) are a class of ubiquitous persistent organic pollutants and they have been associated with declining male fertility. In the present study, we aimed to determine the responsiveness of prosaposin (Psap) expression to PCB exposure. Male C57 mice were exposed to PCB mixture (Aroclor 1254) of environmental related doses by oral gavage. After exposure for 50 days, the expression of Psap was significantly decreased by PCB exposure in epididymides and epydidymal spermatozoa, but not in testis. The Psap abundance in sperm was decreased in a dose-dependent manner. Benchmark dose modeling revealed the 95% lower confidence limit on the benchmark dose (BMDL) and Benchmark Dose (BMD) for Psap reduction were 1.25 and 8.89 µg/kg Aroclor 1254, and for sperm motility reduction were 11.85 and 61.9 µg/kg Aroclor 1254. The depressed Psap level also showed a significant correlation (P<0.01, r=-0.531) with PCB accumulation in liver. In men with detectable PCB exposure in semen, Psap expression in sperm was significantly decreased whereas the semen parameters were unaffected. Linear regression showed that a negative association between total PCB level in seminal plasma and Psap level in ejaculated spermatozoa (P<0.05, r=-0.396). In conclusion, our data suggested that the abundance of Psap in sperm sample may be a sensitive endpoint to predict PCB exposure.

Does prolonged pituitary down-regulation with gonadotropin-releasing hormone agonist improve the live-birth rate in in vitro fertilization treatment?

OBJECTIVE: To evaluate the effects of a prolonged duration of gonadotropin-releasing hormone agonist (GnRH-a) in pituitary down-regulation for controlled ovarian hyperstimulation (COH) on the live-birth rate in nonendometriotic women undergoing in vitro fertilization and embryo transfer (IVF-ET). DESIGN: Retrospective cohort study. SETTING: University-affiliated hospital. PATIENT(S): Normogonadotropic women undergoing IVF. INTERVENTION(S): Three hundred seventy-eight patients receiving a prolonged pituitary down-regulation with GnRH-a before ovarian stimulation and 422 patients receiving a GnRH-a long protocol. MAIN OUTCOME MEASURE(S): Live-birth rate per fresh ET. RESULT(S): In comparison with the long protocol, the prolonged down-regulation protocol required a higher total dose of gonadotropins. A lower serum luteinizing hormone (LH) level on the starting day of gonadotropin and the day of human chorionic gonadotropin (hCG) and a fewer number of oocytes and embryos were observed in the prolonged down-regulation protocol. However, the duration of stimulation and number of high-quality embryos were comparable between the two groups. A statistically significantly higher implantation rate (50.27% vs. 39.69%), clinical pregnancy rate (64.02% vs. 56.87%) and live-birth rate per fresh transfer cycle (55.56% vs. 45.73%) were observed in the prolonged protocol. CONCLUSION(S): Prolonged down-regulation in a GnRH-a protocol might increase the live-birth rates in normogonadotropic women.

Differential expression and anti-oxidant function of glutathione peroxidase 3 in mouse uterus during decidualization.

Glutathione peroxidase 3 (GPX3) is an important member of antioxidant enzymes for reducing reactive oxygen species and maintaining the oxygen balance. Gpx3 mRNA is strongly expressed in decidual cells from days 5 to 8 of pregnancy. After pregnant mice are treated with GPX inhibitor for 3 days, pregnancy rate is significantly reduced. Progesterone stimulates Gpx3 expression through PR/HIF1α in mouse endometrial stromal cells. In the decidua, the high level of GPX3 expression is closely associated with the reduction of hydrogen peroxide (H2O2). Based on our data, GPX3 may play a major role in reducing H2O2 during decidualization.

Mutant ZP1 in familial infertility.

The human zona pellucida is composed of four glycoproteins (ZP1, ZP2, ZP3, and ZP4) and has an important role in reproduction. Here we describe a form of infertility with an autosomal recessive mode of inheritance, characterized by abnormal eggs that lack a zona pellucida. We identified a homozygous frameshift mutation in ZP1 in six family members. In vitro studies showed that defective ZP1 proteins and normal ZP3 proteins colocalized throughout the cells and were not expressed at the cell surface, suggesting that the aberrant ZP1 results in the sequestration of ZP3 in the cytoplasm, thereby preventing the formation of the zona pellucida around the oocyte.

The mesenchymal-epithelial transition during in vitro decidualization.

The epithelial-mesenchymal transition plays a critical role in embryonic development, cancer progression, and metastasis. Decidualization is the process by which the fibroblast-like endometrial stromal cells differentiate into polygonal epithelial-like cells. However, it is still unclear whether mesenchymal-epithelial transition (MET) occurs during decidualization. The aim of this study was to examine whether decidualization causes the downregulation of some mesenchymal markers and upregulation of some epithelial markers in cultured uterine stromal cells. We showed that decidualization causes the downregulation of snail and vimentin expression, and upregulation of E-cadherin and cytokeratin expression. During in vitro decidualization, cultured stromal cells lose elongated shape and show epithelium-like characteristics. Our data suggest that the process of MET may exist during decidualization.

Seminal plasma metals concentration with respect to semen quality.

The aim of the current study was to assess relationships between multiple metals burden in human seminal plasma and semen quality parameters. Levels of five metals (lead, manganese, copper, arsenic, and selenium) in human seminal plasma were determined by inductively coupled plasma mass spectrometry (ICP-MS), and the correlations between the metal concentrations and semen parameters (sperm concentration, sperm motility rate, and sperm morphology) were analyzed. The activities of acid phosphatase (ACP) and of α-glucosidase in human seminal plasma were also determined. Of the 100 subjects, 21 had fertility problems according to the World Health Organization criteria and were designated as "abnormal group." Significant inverse correlations were found between the concentrations of Cu, As, Pb, and the sperm concentrations (r (Cu) = -0.312, P (Cu) = 0.029; r (As) = -0.328, P (As) = 0.021; r (Pb) = -0.377, P (Pb) = 0.008). Moreover, the Cu, Mn, and Se concentrations were significantly higher in the abnormal group than that in the normal group (P (Cu) = 0.024, P (Mn) = 0.002, P (Se) = 0.002). The ACP activity was significantly higher in the normal group than that in the abnormal group (P = 0.021). We also found a significantly negative correlation between α-glucosidase activity and the levels of As (r = -0.367, P = 0.023). These findings provide evidence for relationships between human semen quality and metal exposures. These relationships are consistent with animal data, but additional human and mechanistic studies are needed.

Expression and function of fatty acid-binding protein 4 during mouse decidualization.

Fatty acid-binding protein 4 (Fabp4) is highly expressed in the secondary decidual zone of mouse decidua and deciduoma and stromal cells under in vitro decidualization. Dtprp, a well-known marker of in vitro decidualization, is diminished by small interfering RNA against Fabp4 and FABP4 inhibitor and stimulated through Fabp4 overexpression.

Genome-wide identification of micro-ribonucleic acids associated with human endometrial receptivity in natural and stimulated cycles by deep sequencing.

OBJECTIVE: To identify microRNAs (miRNAs) associated with endometrial receptivity. DESIGN: Observational study. SETTING: Medical center. PATIENT(S): Healthy, regularly cycling women undergoing IVF treatment. INTERVENTION(S): Gonadotropin stimulation and endometrial biopsy. MAIN OUTCOME MEASURE(S): Quantification of miRNA expression profiles by deep sequencing. RESULT(S): The miRNA expression profiles in human endometrium on days LH+2 and LH+7 (LH = 0 is the day of the LH surge) in natural cycles as well as on days hCG+4 and hCG+7 (hCG = 0 is the day of hCG injection) in stimulated cycles were determined by deep sequencing. In natural cycles, there were 20 significantly changed miRNAs in human endometrium on LH+7 compared with LH+2. These miRNAs were predicted to target a large set of genes with different functions, including cell cycle, transport, cell adhesion, cell death, and metabolism. In stimulated cycles, 22 miRNAs were significantly dysregulated on hCG+7 in comparison with LH+7, 11 of which exhibited putative estrogen response elements or P response elements in the promoters. Additionally, unsupervised hierarchical clustering analysis demonstrated that the miRNA expression profile on hCG+4 was similar to that on LH+7, suggesting that ovarian stimulation may alter the window of endometrial receptivity. CONCLUSION(S): MiRNAs may be novel biomarkers for human endometrial receptivity and may help optimize the protocol for IVF treatment.